expression levels of the UR were functional in T cell activation as defined 
by their ability to initiate proliferation and cytolytic activity in response to 
target cells expressing very low levels of HIV antigen. Importantly, the 
CD8+ UR+ T cell population exhibits highly efficient cytolytic activity 
against CD4+ T cells infected with HIV in an antigen specific MHC- 
unrestricted manner. 
The CD4-UR initiate levels of cytolytic activity against 293env target cells, 
exhibiting specific lysis at E;T ratios as low as 0.3:1. Subsequent studies 
were designed to evaluate the effectiveness with which the CD8+ UR+ T 
cell populations were able to mount a cytolytic response against target cell 
types more closely resembling those encountered in an HIV-infected 
individual, namely HIV-infected CD4-I- T cells. The CD4-1- T cell line 
CEM is readily infcctable by the laboratory viral isolate HIV IIIB. When 
the CEM. IIIB cell population were used as targets in a cytotoxicity assay, 
the results obtained were even more dramatic than had been observed with 
the 293env target cells: E:T ratios as low as 0.075:1 resulted in specific 
lysis of the virally infected population, maximal lysis occurring at E:T 
ratios as low as 2.5 -5.0:1 with levels of specific lysis approximately 90% 
above background. The explanation for the increased activity of the UR-i- 
CD8+ T cells against CEM. IIIB compared to 293env, may simply reflect 
the dramatic difference in surface antigen levels observed between the two 
target cells, but also may involve cell-specific factors differing between the 
target CEM T cells and 293 epithelial cells. 
In addition, CD8+ UR+ T cell populations were evaluated for their ability 
to proliferate in response to 293 target cells expressing HIV env. 
Redirected UR+ T cells were incubated with mitomycin C-treated 293. env 
or control 293. neo cells for 3 days, and 3H-Thymidine incorporation was 
measured. In parallel, the CD8+ UR-i- T cell population was stimulated 
with the CD3-specific antibody OKT3 in order compare the proliferative 
response to that obtained by highly efficient stimulation of the native T cell 
receptor by antibody cross-linking. The CD4-UR+ cells proliferate 
extensively in response to stimulation with gpl20 or gp41 respectively 
expressed on the surface of 293env cells. The proliferative response of the 
modified T cell population to stimulation through the UR with surface viral 
antigen is comparable to that obtained by cross-linking the native T cell 
receptor with specific antibody. 
The experimental design outlined here is directly applicable to the clinical 
setting, involving purification of PBMC-derived CD8+ T cells from the 
blood of human donors, introduction of the UR gene via retroviral 
transduction, purification of transduced UR+ T cells, expansion, and 
reinfusion into the patient. The transduced T cell populations produced in 
this manner express high levels of anti-HIV UR, and are able to mount 
highly efficient effector T cell responses such as cytokine secretion, 
proliferation, and cytolytic activity upon interaction with target cells 
expressing extremely low levels of surface HIV antigen. This approach 
can be used to obtain large populations of redirected T cells in a matter of 
weeks. 
The relative affinity of the CD4 receptor for nonpolymorphic sites on HLA 
class II molecules is thought to be very low in absence of additional 
stabilizing TCR-HLA interactions. However, the potential for CD4-UR 
modified T cell populations generated to initiate a cytolytic response against 
Recombinant DNA Research, Volume 19 
[135] 
